Abstract
Background/Aim: MicroRNAs (miRs) are non-coding RNA molecules regulating diverse cellular processes essential in carcinogenesis. Little is known regarding miRs in head and neck squamous cell cancer (HNSCC). The aim of the present study was to investigate miRs in relation to the clinico pathological features of site-specific HNSCC. Materials and Methods: The study comprised of 51 patients with HNSCC (23 oropharyngeal, 24 laryngeal and 4 hypopharyngeal carcinomas). Total RNA was extracted from tumor tissue and normal squamous epithelium using the miRNeasy FFPE Kit. A quantitative estimation of let-7a, miR-21, miR-200c, miR-34a, miR-375 was performed by a real-time poly merase chain reaction (PCR) method using the TagMan® MicroRNA assay. Additionally, p16 expression was detected by immuno histo chemistry. Results: Significant differences of let-7a, miR-200c, miR-34a levels between oropharyngeal and laryngeal cancers were found (p<0.05). Compared to non-neoplastic tissues, miR-21, miR-200c, miR-34a were up-regulated and miR-375 was down-regulated in tumors of all sites. MiR-34a tumor levels significantly correlated with oropharyngeal origin (p=0.0284) and p16 positivity (p=0.0218). Conclusion: The microRNA profile seems to play a potential role in the pathobiology of oropharyngeal and laryngeal HNSCC. Up-regulation of miR34a in p16-positive oropharyngeal cancer has not been so far described and additional studies are warranted.
- MicroRNA
- let-7a
- miR-21
- miR-200c
- miR-34a
- miR-375
- head and neck squamous cell carcinoma
- oropharyngeal
- laryngeal
- hypopharyngeal cancer
The head and neck squamous cell cancer (HNSCC) represents a broad scale of tumors from the oral cavity to larynx (1). HNSCC of different anatomical sites seems to be associated with different etiopathogenesis, molecular characteristics and clinical outcomes, despite the same histological type. The majority of previous studies on HNSCC were performed irrespective of tumor location and published results were not site-specific. Detailed knowledge of the mole cular basis of these tumors is, thus, required and new HNSCC biomarkers are warranted.
Nowadays, there is an increasing interest in the role of microRNAs (also known as miRNAs or miRs) in physio logical and pathological cell processes, which bring new insights to cancer pathobiology. MicroRNAs are newly-recognized, non-coding, regulatory RNA molecules, 18-25 nucleotides in length. Their biogenesis is a multi-step process under the control of several enzymes and enzymatic complexes (Figure 1). Briefly, this process starts in the nucleus, where the primary-micro RNA (pri-micro RNA) with long nucleotide sequence is produced. During the next step, the hairpin-shaped pri-micro RNA enters a complex consisting of the enzyme Drosha and an essential cofactor Pasha to be processed into pre-microRNA and then transported to the cytoplasm. Enzymes Dicer and helicase are responsible for shortening of double stranded RNA and subsequently unwinding of this duplex into two mature microRNAs. They are incorporated into the RNA-induced silencing complex (RISC), which regulates the final effect of microRNA. For detailed description of micro RNA biogenesis, we recommend earlier reviews (2-4). Expression of microRNA is tissue-specific and each alteration of tissue microRNA profile is associated with distinct disease-status. MicroRNAs participate in post-transcriptional regulation of gene expression to control development and maintain diverse cellular processes, including proliferation, apoptosis, senescence, differentiation, motility and morphogenesis. Each microRNA can regulate a considerable number of genes downstream by targeting many messenger RNA (mRNA) transcripts. One mRNA may be influenced by more types of microRNA. The final effect of this interaction is mRNA cleavage or translational repression (depending on the degree of complementarity with the target mRNA) with simultaneous quick degradation of microRNA. Thus, microRNA can control more than one-third of the protein-coding genes in the human genome inclusive of genes coding for transcription factors and RNA regulating proteins (5). Transregulation represents subsequent functional effects of microRNAs that may alter the levels of other RNAs (5).
MicroRNAs are involved in many diseases, both non-neoplastic and cancers. Their link to cancer is not surprising taking into consideration that microRNAs are involved in many processes essential in carcinogenesis and cancer progression (e.g. proliferation, migration, apoptosis). Micro-RNA dysregulation is attributed to germ-line or soma tic mutations of microRNA genes or epigenetic changes (6, 7). Two principal effects of microRNAs in human cancer have been described: oncogenic and tumor-suppressor. The former is linked to negative regulation of tumor suppressor genes, while the latter is mediated by translational repression of oncogenes (8, 9). However, a dual effect of some microRNAs, both onco genic and tumor suppressing, has also been described (10).
More than 2,500 mature microRNAs have been identified in humans (see miRBase Sequence Database, Release 21, available online at www.miRbase.org) with different roles in cancerogenesis (11). In our study, we focused on 5 selected microRNAs (in Table I) with potentially crucial roles in the pathobiology of site-specific HNSCC: (i) Let-7a belongs to the large let-7 family. The let-7a precursor was the first microRNA identified from the study of developmental timing in Caenorhabditis elegans. The ubiquitously expressed let-7/miR-98 was one of the first mammalian microRNAs to be described. The let-7 family is often present in multiple copies in the genome and isoforms with slightly different sequences are indicated by a letter (12). Along with the general function to promote the differentiation of cells, its tumor suppressor role by targeting multiple oncogenes, namely RAS, in various human cancers, including head and neck cancer, was recently described (13). Moreover, its repressive role in cancer stem cells with stem-like properties ablation was also recently described (14). (ii) MicroRNA-21 is one of the first microRNAs identified in a number of cancers also having an important role as an oncogene (15). MiR-21 has been proven to be up-regulated in a wide variety of malignant tumors, namely in human glioblastomas, breast, colon, lung, pancreas, prostate cancer and in head and neck squamous cell carcinoma as well (16, 17). MiR-21 over-expression was significantly associated with worse outcome promoting cell proliferation (16, 18). Recently, miR-21 was introduced as a promising diagnostic and prognostic bio marker and thera peutic target as well (17, 19-21). (iii) MicroRNA-200c is one of the five members of microRNA-200 family that regulates the epithelial-mesenchymal transition (EMT) by targeting EMT-related gene expression. MiR-200c exhibits tumor suppressive properties. Its down-regulation promotes EMT-facilitating tumor cell invasion and progression of cancer, while up-regulation induces mesenchymal-epithelial transition (22, 23). (iv) MicroRNA-34a, like one of the three members of miR-34 family, is characterized by its tumor suppressor action that induces apoptosis, cell-cycle arrest, senescence and EMT. The p53 family has been described as one of the principal inducers of miR-34a gene expression, although alternative regulatory pathways also exist (24, 25). MiR-34a dysregulation plays an important role in many human cancers; this small molecule is, usually, down-regulated and associated with poor outcome (26). Common causes of miR-34a low expression are deletion, CpG promoter methylation of the MIR-34A gene (RNA gene) on chromosome 1 or the alteration of p53. This shows the important therapeutic potential of miR-34a as an anticancer drug (27). However, little is known regarding the role and expression status of miR-34a in head and neck squamous cell carcinomas. (v) MicroRNA-375 is typically expressed in the pancreas and pituitary gland. It has a great impact on development and endocrine function of the pancreas. MiR-375 has been implicated in a number of different cancers being down-regulated in cancer cells when compared to adjacent non-neoplastic tissues. Since there exist several mole cular targets and different pathways of miR-375 regulation, it is necessary to investigate the expression pattern in specific cancers (28). Generally, miR-375 acts as tumor suppressor in human cancer, although an oncogenic function in breast cancer has been described (29).
Therefore, the aim of the present study was to investigate the profile of the above-mentioned microRNAs in HNSCC of different anatomical sites in relation to etiological factors and other clinicopathological features of these tumors.
Patients and Methods
Patients. The study patient population comprised of 51 patients with HNSCC treated from July 2009 to December 2012 at the Department of Otorhinolaryngology and Head and Neck Surgery, Faculty Hospital in Hradec Kralove, Czech Republic. The HNSCC groups included three sub-groups represented by histologically-verified squamous carcinomas of the oropharynx (n=23 patients), hypo pharynx (n=4 patients) and larynx (n=24 patients). Patients included in the study were both smokers and non-smokers. Grading of tumors was obtained from pathological reports. Staging of tumors was evaluated according to pTNM Union for International Cancer Control (UICC) pathological staging criteria (30). All participants were informed on the research study and written inform consent was obtained from each patient. This work was approved by the Ethics Committee of the University Hospital in Hradec Kralove. Patients with other malignancies, inflammatory diseases or infections were excluded. Treatment decision-making was based on clinical status of patients and on grading and staging of tumors. All patients underwent surgery with resection of the tumor. The clinicopathological profile of the study populations is shown in Table II.
Tissue samples. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with HNSCC were prepared by standard laboratory technique and stored at room temperature until use. Samples, collected during a 4-year period (from 2009 to 2012), were processed at the Fingerland Institute of Pathology at the University Hospital in Hradec Kralove. Four-μm-thick paraffin sections were stained with hematoxylin and eosin and microscopically evaluated to ascertain regions suitable for macrodissection. Minimally, two FFPE tissue samples from each patient were histologically analyzed by a pathologist (M.L.) in order to find sites with cancer cells and sites of adjacent non-cancerous epithelial tissue. All selected areas destined for microRNA analysis were manually highlighted on hematoxylin and eosin stained slides. Thereafter, FFPE tissue samples were cut into 50 μm sections and parts of these sections, corresponding with previously highlighted areas, were separated. A total tissue area of approximately one cm2 was used for RNA extraction. The estimation was performed in 51 paired (tumor and control) tissue samples of HNSCC.
RNA isolation. RNA was extracted from 50 μm FFPE sections following macrodissection of HNSCC tumor tissue and adjacent non-cancerous tissue using the miRNeasy FFPE Kit (Qiagen, Hilden, Germany).
Real-time quantitative PCR (qRT-PCR). A quantitative estimation of let-7a, miR-21, miR-200c, miR-34a, miR-375 and U6snRNA was performed by a qRT-PCR method using TaqMan® MicroRNA Assays (supplier, address). A two-step protocol requires reverse transcription with a miRNA-specific primer followed by a real-time PCR with TaqMan® probes (give more details and/or reference). The assays target only mature microRNAs, not their precursors. We used RNU6B (U6snRNA) as a normalizer. Each sample was assessed twice in parallel. The Ct values were corrected using calibrators for the elimination of differences between runs of the Stratagene Mx3000P Real-Time PCR apparatus (Agilent Technologies, Santa Clara, CA, USA). For the statistical analysis, we used the ddCt approach (2-Δ Δ CT algorithm; Applied Bio systems, Foster City, CA, USA).
Determination of p16 status. In our Hospital (Faculty Hospital in Hradec Kralove, Czech Republic), p16INK4a (hereafter denoted as p16) expression is routinely evaluated by immunohistochemistry within the field of activity of the histological examination of every HNSCC. Therefore, p16 status data are available as they were retrieved from hospital records. Immunohistochemistry was performed with the CINtec® Histology Kit (Roche mtm laboratories AG, Heidelberg, Germany). The p16 immunostaining was scored as follows: negative (0-50% tumor cells-nuclei and/or cytoplasmstained); positive (51-100% tumor cells-nuclei and/or cytoplasm-stained).
Statistical analysis. Data analysis was performed using the SAS 8.02 software (SAS Institute Inc., Cary, NC, USA). The statistical results were calculated by the Wilcoxon non-parametric two sample test. Values of p lower than 0.05 were considered to be statistically significant in all analyses.
Results
The clinicopathological data of the HNSCC patients in the studied cohort are summarized in Table II. The expression levels of let-7a, miR-21, miR-200c, miR-34a and miR-375 were compared between site-specific sub-groups (oropharyngeal, hypopharyngeal and laryngeal) of HNSCC and between neoplastic and non-neoplastic tissues of the same patient. Comparison of the expression ratio of studied microRNAs with the clinicopathological and etiological characteristics of tumors was also performed.
Comparison of expression status of microRNAs between neoplastic and non-neoplastic tissues and between site-specific tumor subgroups. Comparing the expression values of the studied microRNAs between tumorous (irrespective to tumor site) and normal tissues of the same patient, we found statistically significant differences in miR-21 (p=0.0001) and miR-200 (p=0.0335), as well as in miR-375 (p=0.0020) expression values. The first two mentioned microRNAs were up-regulated, while the last one was down-regulated in the tumor tissue. All measured values of the expression of the studied microRNAs in tumorous and healthy tissues are summarized in Table III. MiR-34a (p=0.0155) was significantly up-regulated in oropharyngeal carcinomas when only comparing neoplastic and normal epithelial cells.
Statistically significant differences were recorded in the expression of let-7a (p=0.0242) and miR-200c (p=0.0378) between oropharyngeal and laryngeal cancers, in the expres sion value of miR-34a between oropharyngeal and laryngeal (Table IV), oropharyngeal and hypopharyngeal, as well as laryngeal and hypopharyngeal carcinomas (p=0.0178; p=0.0355; p=0.0317, respectively). In all cases, the highest expression levels of miR-34a were found in oropharyngeal cancers.
Expression tumor status of microRNAs in relation to clinicopathological features of HNSCC patients. No corre lation was observed between microRNA tumor expression and traditional clinicopathological factors, like sex and age. Furthermore, taking all HNSCC tumors together (irrespective to tumor site), statistically significant differences were found in tumor expression levels of let-7a and miR-200c between low stage (I-II) and high stage (III-IV) cancers (p=0.0204; p=0.0492, respectively), whereas higher tumor microRNA expression was associated with high HNSCC stage in both biomarkers. We also recorded significantly higher expression of miR-200c (p=0.0092) in less differentiated HNSCC (G3) and miR-375 (p=0.0231) in lymph node-positive (N1-3) laryngeal carcinomas. There were no significant differences in the miR-21, miR-34a, let-7a and miR-375 or miR-200c expression between low-grade and high-grade tumors, cancer lymph node status (N0 versus N1-3) and non-smoker and smoker patient groups. However, a significantly higher miR-21 (p=0.0005) and lower miR-375 (p=0.0010) expression in tumor than in healthy tissues was found in smokers but not in non-smokers.
HNSCC microRNAs expression in relation to p16 status. Comparing the p16 expression in site-specific head and neck tumors, p16 positivity has a statistically significant relationship to oropharyngeal cancer (p=0.0001) but not to hypopharyngeal and laryngeal tumors. Moreover, oropha ryn geal carcinomas had a significant relationship between p16 expression and non-smoking (p=0.0428) and response to therapy (p=0.0018). Taking all HNSCC cases together, a statistically significant p16 tumor positivity was proven in patients with lymph node metastases (p=0.0173), in non-smokers (p=0.0001) and in tumors without recurrence/ persistence (p=0.0294). With respect to tumor site, the afore mentioned significant relationship was confirmed in oropha ryn geal tumors only (p=0.0018).
Down-regulation of miR-375 in HNSCC neoplastic cells was significantly associated with p16 tumor negativity (p=0.0038). Comparing all the site-specific p16 negative cancer sub-groups, the highest statistically significant miR-375 expression was found in oropharyngeal tumors (p=0.0441).
MiR-34a was significantly up-regulated in oropharyngeal tumors (p=0.0155) and in p16 positive carcinomas (p=0.0267). MiR-21, let-7a, miR-375 and miR-200c levels did not significantly correlate with p16 expression.
Discussion
HNSCCs represent about 6% of all cancer cases worldwide, with the majority being oropharyngeal and laryngeal squamous carcinomas (31). Smoking, alcohol abuse and human papillomavirus (HPV) infection have been acknowledged by the International Agency for Research against Cancer (IARC) as basic etiological factors of HNSCC (32). Generally, HNSCC is considered to be an aggressive neoplasm with unfavorable prognosis, despite the improvement of therapy in the last decades, and is often studied together as a single disease. However, behavior diversity of these tumors in different head and neck locations probably reflects miscellaneous pathways of cancerogenesis and specific intrinsic tumor properties, which are known from clinical practice (33). The locoregional lymph node metastasis is an important prognostic factor for these cancers. Moreover, many node-negative (N0) classified HNSCCs patients harbor occult neck node metastases (34). HNSCC remains difficult to be clinically managed. Therefore, a better understanding of the molecular pathobiology of head and neck squamous cell cancer is required. MicroRNAs represent new molecules regulating gene expression at the post-transcriptional level by the translational repression and/or degradation of target mRNA (35). MicroRNAs have distinct expression profiles in multiple pathophysiological conditions, including cancer. A number of studies on microRNA expression profiles of breast, prostate, colorectal, brain and other cancers have been so far published (18, 21, 35-37). However, little is known regarding the role of microRNAs in head and neck squamous carcinomas mainly with respect to their site of origin. Therefore, further studies of microRNA expression profile in site-specific HNSCC with potential clinical efficacy for diagnosis, pro gnosis and treatment seem to be fully substantiated (38). Certain studies have focused on microRNA expression profile of head and neck cancer (35, 39). The results seem to be inconclusive as various microRNAs were studied under different methods in different samples/cell lines and in different clinicopathological conditions. Although a vast number of de-regulated microRNAs have been reported, only a limited number of microRNAs share a common type of de-regulation pointing out to their important role in head and neck cancerogenesis and their potential prognostic function (35). In the study of Avissar et al. (2009), comparing neoplastic and normal tissues, it was shown that miR-21, miR-221, miR-18 were up-regulated in HNSCC tumors, whereas miR-375 was down-regulated (40). Moreover, the expression ratio of miR-221/miR-375 was introduced to be a promising diagnostic marker in distinguishing tumor from normal tissue. The same study revealed significant discrepancies in microRNA expression between cell lines and tumor tissues warning about limited utility of cell lines as a model system for the identification of clinically relevant microRNA biomarkers (40). MicroRNAs tested in the present study have been previously described to play crucial role(s) in carcinogenesis either in HNSCC or other cancer types.
In the present study, by comparing tumors to healthy tissues, we found significant differences in miR-21, miR-200c and miR-375 expression. Up-regulation of miR-21, a repeatedly confirmed finding, was first revealed in HNSCC (39-42). Down-regulation of miR-375 (being under-expressed by 9-fold compared to normal tissue in the present study) has also been reported in previous studies (40). Little is known about the deregulation of miR-200c in head and neck cancer, although its tumor-suppressive role in renal cell carcinoma, prostate, bladder, breast, pancreatic and gastric cancers has been described (35). Up-regulation of miR-200c was proven to inhibit the cancer stem cell-like properties and support MET by targeting the ZEB1/ZEB2, BMI-1 and SOX2 genes (23, 35). However, the role of miR200c has not been yet reported in the regulation of tumorigenicity and clinical behavior of HNSCC. MiR200c was significantly up-regulated and a considerable correlation of its over-expres sion with less differentiated cancers, but not with high-stage cancers, was also revealed in our recent study. However, this over-expression, with respect to its tumor-suppressive function, could be expected to be related to low-grade cancer. The small number of cases studied and incomplete elucidation of miR-200c interaction are shortcomings for better interpretation of these results.
Concerning the evaluation of microRNA expression profiles in different site-specific sub-groups of HNSCC and comparing them with each other, we found that let-7a, miR-200c and miR-34a revealed significant differences between oropharyngeal and laryngeal tumors. Surprisingly, miR-34a was significantly more up-regulated in oropharyngeal carci no mas. We are aware of only one study by Hui et al. that focused on microRNA profiling in subgroups of HNSCC (43). However, the authors did not find any differences in microRNAs expression of the oropharynx, the larynx and the hypopharynx. Besides miR-21 and miR-375, the other evaluated microRNAs were different from those explored in our study (43).
Taking all HNSCC cases together, we found significant differences between let-7a, as well as miR-200c expression, and stage of tumors. In laryngeal carcinomas, only miR-200c expression was higher and significantly associated with high stage of cancer, whereas miR-375 was associated with laryngeal lymph node-positive carcinoma. Generally used prognostic parameters, like disease free interval (DFI) and/or overall survival (OS), could not be evaluated in relation to microRNA expression levels due to relatively short follow-up in our study. Nevertheless, the monitoring of let-7a, miR-200c and miR-375 seems to have some potential prognostic significance. The prognostic role of selected microRNAs has been evaluated in HNSCC by several authors but the results seem to be heterogeneous and inconclusive for clinical practice (44).
Cancer pathobiology and the molecular differences between the anatomical locations and tumors of different etiology (viral, smoking or alcohol-induced) are important for developing new molecular targeted strategies. MicroRNA patterns are tissue-specific and may be influenced by the diversity of environmental factors.
Smoking-related cancers are the common object of microRNA studies, namely lung cancer. There exist different pathways of microRNA dysregulation: modification of microRNA gene expression followed by mutation or epigenetic regulation of distinct genes or by alteration of tumor suppressor or oncogenic microRNA function (45). Smoke-induced cancers show usually a bimodal microRNA expression profile with an initial down-regulation and a final up-regulation in advanced stages of cancer. In our study, we revealed significantly higher miR-21 (p=0.0005) and lower miR-375 (p=0.0010) expression in the tumor of smokers than in healthy tissues.
Alcohol represents one of the risk factors of HNSCC pathobiology. Alcohol has been described to contribute to dysregulation of microRNAs by several mechanisms, like the irritation of inflammation, interference with the absorption of folate and/or its degradation into carcinogenic acetaldehyde. Expression of miR-375 has been shown to increase with alcohol consumption in HNSCC (46). Our research group did not reveal any association between the studied microRNAs and alcohol use. However, it is necessary to take into consideration that clinical data in relation to drinking can be subject of variability.
The role of HPV/p16 in the regulation of microRNA expression in head and neck cancers is largely unknown (47). P16 positivity of HNSCC is considered to be a surrogate marker of HPV etiology. However, other causes of p16 expression are also suggested. Clinical features and prognosis of HNSCC are greatly influenced by the HPV/p16 status, which is favorable in p16-positive tumors (48). The function of p16 seems to be more complex and differs between tumors of various locations. Therefore, the study of p16 role in tumors, in relation to its prognostic and therapeutic consequences, should be site-specific (49, 50). Oncogene-induced senescence represents a barrier against tumorigenesis. P16 is known as a key regulator of cellular senescence in cooperation with senescence-associated micro-RNAs. Namely, accumulation of miR-34a has been described during senescence in a range of cell types (25, 49, 51). The tumor-suppressive function of miR-34a has been reported in multiple cancers. Tumor suppressive effects consist of deregulation of oncogene expression and are mediated by changes in a number of target mRNAs, including MYC, cyclin B1 (CCNB1), BCL-2, E2F3 and surivivin (BIRC5) (11, 25, 52, 53). In the majority of cancers, miR-34 is down-regulated and associated with tumor progression (11, 26). Ogawa et al. described miR-34 down-regulation to be associated with cis-diamminedichloroplatinum (CDDP) resistance of sinonasal squamous cell carcinoma (54). Contrary to the majority of cancers, miR-34 was overexpressed in our study, namely in p16-positive oropharyngeal carcinomas. This fact could be responsible for more favourable prognosis of these tumors described earlier (48). Unfortunately, the number of cases in our pilot study was limited. Therefore the studies of larger cohort of p16-positive oropharyngeal carcinomas are required to confirm miR-34a overexpression. The elucidation of its underlying pathobiological mechanisms and possible association miR-34a up-regulation with oncogene-induced senescence and clinical consequences are warranted.
Published articles on head and neck microRNA de-regulations are still scarce but expanding. However, since different con clu sions have been formulated in reported studies, the use of microRNA profile of HNSCC in routine clinical practice cannot be currently asserted (42, 47, 55). Our study was performed using FFPE tissue samples. Although FFPE samples do not seem to be useful for mRNA, as well as DNA analysis due to their degradation during the fixation process and deterioration over time, the isolation of small-sized microRNA from these samples seems to be fully substantiated as micro-RNAs are known to be more stable (56). Moreover, the study of microRNAs in HNSCC should be more complex by taking many exogenous and endogenous aspects, including tumor location, into consideration. Nevertheless, based on our pilot study and the literary knowledge, we are convinced that microRNA profiling of HNSCC is behind the diagnostic, prognostic and therapeutic potential of these tumors. Therefore, microRNAs seem to potentially extend the family of tumor biomarkers in the future.
Conclusion
This study revealed that certain microRNA profiles are de-regulated in head and neck cancer (up-regulation of miR-21, miR-200c and miR-34a; down-regulation of miR-375) playing a potential role in the pathobiology of HNSCC. Significant differences of microRNA expression (let-7a, miR34a) between oropharyngeal and laryngeal cancers support the hypothesis of site-specific cancerogenesis in HNSCC. Up-regulation of miR34a expression in p16-posi tive oropha ryn geal carcinomas has not been so far described. The role of miR-34a in carcinogenesis is more complex and remains to be elucidated. Additional microRNA expression studies in site-specific HNSCC in relation to prognosis, smoking and HPV infection/p16 status, respectively, are warranted.
Acknowledgments
This study was supported by a research project of Charles University in Prague, Czech Republic (SVV No.260050), by the Charles University Research Fund (projects PRVOUK No. P36 and P37) and by a research project of the Ministry of Health, Czech Republic, No. 00669806 (Faculty Hospital Plzen).
Footnotes
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Conflicts of Interest
The Authors declare that there exist no conflicts of interest regarding this work.
- Received December 15, 2014.
- Revision received January 15, 2015.
- Accepted January 21, 2015.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved